Proton activated atomic medicine

ABSTRACT

The present application provides compositions and methods for preparing and using “heavy” nucleotide derivatives of thymidine or uridine by replacing the oxygen atom attached to one or more of positions with non-radioactive oxygen-18 (18O), administering it to a subject to target a tumor including incorporation into tumor cell DNA, and then treating the tumor with proton beam therapy to transmutate the 18O to 18F, resulting in a break of the new fluorine-phosphorous bond. This chemical event destabilizes ribose-phosphate DNA back-bone and base pairing thus produce single- and double strand breaks, clusters lesions that can lead to irreparable DNA damage and enhanced tumor cell killing. The atomic, chemical, and physical aspects result in the use of lower radiation doses and significantly alter acute and late morbidity of radiotherapy. Heavy thymidine and heavy uridine derivatives labeled with 18O have been made and tested.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application Ser. No. 62/523,349 filed Jun. 22, 2017, thedisclosure of which is incorporated by reference in its entirety herein.

BACKGROUND

Radiation therapy, whether used alone or combined with other treatments,is an important treatment for many cancers. More than half of all cancerpatients receive one or more courses of radiation therapy as part oftheir treatment. However, in radiation therapy, large amounts of energyare directed at cancer cells to disrupt growth and x-rays are the typeof energy found in conventional radiation therapy. Unfortunately, bothhealthy and cancerous cells are affected by radiation, so the goal is toradiate only the targeted cancer cells.

Both proton therapy and traditional radiation treat malignancies thesame way: by inhibiting the growth of cancer cells. Proton beam therapyreduces many of the issues associated with conventional radiationtherapy. Protons carry the energy in proton therapy and the protons areraised to a high energy level using a particle accelerator. An advantageof proton therapy is that proton beams stop after releasing their energywithin a target and the beam can be more finely controlled, allowing forhigher doses of radiation to be used because the tumor can be morespecifically targeted. Thus, surrounding healthy tissue is less affectedthan when x-rays are used. Protecting healthy tissue is particularlyimportant when the patient is a child. Children can have the greatestlong-term harm from conventional radiation therapy since their organsare still developing. Delayed effects of X-ray therapy in children caninclude growth problems, hearing and vision loss, radiation-inducedcancers, and heart disease.

Proton therapy is typically used for tumors that have not spread toother parts of the body and is effective in treating many types ofcancer. Proton therapy may be most valuable in the treatment of tumorsincluding, but not limited to, brain, breast, bone, gastrointestinaltract, prostate, and pediatric cancers.

Because there are not many cancer centers utilizing proton beam therapy,its full potential has not yet been realized.

There is a long felt need in the art for compositions and methods usefulfor diagnosing and treating diseases and disorders encompassingexcessive cell proliferation, particularly cancer. The present inventionsatisfies these needs.

SUMMARY OF THE INVENTION

The present application discloses novel purines and pyrimidines in whichoxygen atoms are replaced with heavy oxygen and are used in proton beamtherapy and in diagnostics. The predominant literature on ¹⁸O deals withits precursor role in the production of diagnostic agents like¹⁸F-deoxyglucose for clinical PET scanning that now can be done withmini-cyclotrons in community hospitals. The literature shows that ¹⁸Oincorporates well into HT analogues (97%); evolving methods with thisisotope currently includes biomarkers for oncology opening thepossibility for the creation of other tagged heavy analogues.

In one aspect, the oxygen atoms attached, for example, at the 3′ or 5′position of the ribose structure are replaced with heavy oxygen(oxygen-18/¹⁸O).

Four different sites of labeling are disclosed herein. In one aspect,the oxygen atoms at both positions are replaced. In another aspect, theoxygen atom at the 2 position on a thymine ring is replaced with ¹⁸O. Inone aspect, at least two oxygen atoms are replaced with ¹⁸O. In oneaspect, all oxygen atoms are replaced with ¹⁸O. In one aspect, heavythymidine is prepared and used.

In one aspect, the present application discloses compositions andmethods useful for making and using non-natural nucleotide derivatives.In one aspect, the non-natural nucleotides are modified with ¹⁸O. In oneaspect, the non-natural derivatives are deuterated. In one aspect, thenon-natural nucleotides are modified with ¹⁸O and are deuterated.

Compounds of the invention are administered to a subject for use as adiagnostic/imaging agent and/or for therapeutic use. In one aspect,after a compound has incorporated into cellular DNA the ¹⁸O targetingagent is activated by proton treatment. In one aspect, the cell is acancer cell. In one aspect, the cell is killed.

In one embodiment, the proton treatment activates the ¹⁸O targetingagent and the ensuing particle-nuclear reaction transmutates ¹⁸O into¹⁸F. In one aspect, a proton beam is used to induce the transmutation.In one aspect, once transmutation occurs the new atomic state results ina chemical break of the fluorine-phosphorous bond. Breaking the bondleads to destabilization of the ribose-phosphate DNA ladder or alteredbased pairing, resulting in, inter alia, altered DNA damage repair andincreased cell death.

It is disclosed herein that a nucleotide can be selectively substitutedat the atomic level using heavy oxygen, that it incorporates into DNA,and it magnifies the dose effect by a significant amount. When combinedwith proton beam therapy there is unexpectedly an even greater increasein sensitivity of cells to the treatment.

In one embodiment, once transmutation occurs, ¹⁸F can be detected andmeasured using PET. ¹⁸F is a useful imaging probe. A tumor being treatedcan be imaged before or after administration of an ¹⁸O labeled compoundof the invention. One of ordinary skill in the art can determine whichtype of imaging to perform. A tumor can also be monitored before andafter treatment using imaging procedures.

The present application discloses derivatives of both thymidine anduridine that have been labeled at one or more positions with heavyoxygen (¹⁸O).

Thymidine has the structure:

In one embodiment, the present application discloses generic formulascomprising structures of useful compounds of the invention. Fourdifferent oxygen sites for substitution with ¹⁸O are disclosed andprovided below for thymidine derivatives. In one aspect, the presentapplication provides compounds selected from the formula consisting of:

wherein:

X is selected from the group consisting of CH₃, halogen, CF3, OH, NH₂,OR, N₃, and CH₂X; and

R is selected from the group consisting of alkyl, alkene, alkyne, andaryl.

Halogens can be F, Cl, Br, or I. When fluorine is used, it can bepresent in a compound as CF, CF₂ or CF₃.

In one embodiment, a compound is deuterated.

Some singly O¹⁸ labeled thymidines of the invention include:

Some thymidines labeled with multiple ¹⁸O molecules include:

Also provided are examples of heavy oxygen 18 labeled 2′-deoxyuridinethymidine (also referred to as 2′-deoxyuridine). The same foursites comprising oxygen can be substituted with ¹⁸O as for thymidine.Singly ¹⁸O labeled uridines, include, but are not limited to:

Some uridines that are multiply labeled with ¹⁸O include:

In one embodiment, the present application provides for deuteratedmodifications to the compounds of the invention.

Proton therapy as disclosed herein when using a compound of theinvention can be used in combination with other treatments such asconventional X-ray radiation, chemotherapy, and surgery. Based on thepresent disclosure, one of ordinary skill in the art can determine if acombination treatment should be used.

The present application provides methods for administering ¹⁸O labeledcompounds to a subject, subjecting the subject to a proton beam andconverting the 18O to radioactive ¹⁸F. Radioactive ¹⁸F can then bedetected and measured in the subject. PET imaging can be used to detecttumors that are present, provide diagnoses, quantify the label present,and can be used in follow up procedures to monitor treatment.

When treating a subject, exposure to a proton beam can be from about 2to about 100 Gy. In one aspect, treatment is about 2, 6, 8, 10, 20, 30,40, 50, 50, 70, 80, 90, 100, 110, 120,130, 140, and 150 Gy. In oneaspect, fractional doses are about 2-10 Gy. In one aspect, total dosesare about 50-150 Gy. In one aspect, proton beam irradiation is with 160MeV protons single fraction. Accelerators used for proton therapytypically produce protons with energies in the range of 70 to 250 MeV.

A treatment regimen may require about five treatments for about 4 toeight weeks. The physician can establish the regimen based on criteriasuch as the type of cancer, its location, and the age, sex, and healthof the subject.

In one embodiment, prior to treatment the tumor is imaged using, forexample, CT to create a virtual model of the tumor.

After administration of an ¹⁸O labeled compound to a subject, it can beused for imaging before being subjected to a proton beam.

In one embodiment, an ¹⁸O labeled compound of the invention is subjectedto a proton beam prior to administration to a subject. In one aspect,the ¹⁸O is transmutated to a radioactive ¹⁸F.

In one embodiment derivatives of thymidine or uridine are made and usedcomprising a ¹⁹F label. In one embodiment, subjecting the derivative toa proton beam converts ¹⁹F to ¹⁸F.

Based on the disclosure provided herein, the present inventionencompasses the beginning of Proton-Activated Atomic Medicine (PAAM)using design science to provide new molecules for diagnosis andtreatment, and is useful as a new radiation therapy paradigm that isbetter than prior proton beam therapy and diagnosis and the use ofconventional irradiation. The compositions and methods encompass:

-   -   transmutation: imaging and chemical consequences;    -   target: atomic sites in DNA and RNA;    -   chemistry achievable at reasonable cost;    -   physiologic doses, non-radioactive, non-toxic;    -   proliferation specific incorporation: cancer greater than normal        tissues; and    -   proton dose distribution will activate cancer and avoid rapidly        proliferating tissues.

In one embodiment, the present application provides compositions andmethods for killing proliferating cancer cells. In one aspect, themethod comprises contacting a proliferating cancer cell with aneffective amount of an ¹⁸O labeled compound of the invention andallowing the compound to incorporate into the DNA of the proliferatingcancer cells, followed by subjecting the proliferating cancer cells toproton beam therapy.

The types of cancer typically subjected to proton beam therapy areencompassed by the methods of the invention and include, but are notlimited to, brain, breast, bone, gastrointestinal tract, prostate, andpediatric tumors and cancer cells.

The present invention encompasses a kit comprising at least one ¹⁸Olabeled compound of the invention, a pharmaceutically acceptablecarrier, an applicator, and an instructional material for the usethereof.

Various aspects and embodiments of the invention are described infurther detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphic representation of the dose of irradiation (x-axis)versus the log survival fraction (y-axis) of log phase U87 humanglioblastoma cells. Three groups were used. The solid line is the resultof treatment with conventional x-rays. There are about 2 logs of cellkill with 10 Gy with x rays. Shown are the survival fractions with 8 Gyof proton irradiation either alone or combined with normal thymidinewhere cells were exposed with a 0.5 micro molar concentration in vitrofor 96 hours prior to proton beam treatment. Below that is shown thesurvival fraction for the same concentration and duration of exposurewith “heavy” thymidine. This molecule is synthesized by replacing the 2position of the aromatic ring of the nitrogenous base with oxygen-18.When these cells are treated with protons, there is a chemicaltransmutation at the atomic level where upon the oxygen-18 atom ischanged into radioactive fluorine-18. This causes a chemical change atthe molecular level and results in greater cell kill. Td- thymidine;HTd-heavy thymidine.

FIG. 2 provides schematically deoxyribonucleotide structures inphosphorylated forms.

FIG. 3 provides a schematic representation of ¹⁸O transmutation and thechemical consequences of the transmutation.

FIG. 4 provides a schematic representation of sites for chemicalmodification of pyrimidine nucleosides and types of modifications thatcan be made at those sites, using 2′ deoxyuridine as a model. (based onFIG. 6 from Wiebe, Brazilian Archives of Biology and Technology, 2007,50:3:445-459).

FIG. 5, comprising FIGS. 5A and 5B, provides schematically a conceptualdepiction of Proton Activated Atomic Medicine. FIG. 5A depicts heavyatom labeled biologically important monomeric entities, transition tooligomeric entities, formation of macromolecules, and the proton beamirradiation causing transmutation and structural changes and leading toaltered function. FIG. 5B illustrates molecular details depicting anucleoside being exposed to a proton beam, atom-transmutation forexample from ¹⁸O to ¹⁸F and then altered structure and function ofbiomolecules.

FIG. 6 demonstrates the synthesis of heavy oxygen 18 labelednucleotides.

FIG. 7 provides examples of heavy oxygen 18 singly labeled thymidinederivatives of the invention, comprising a site for various substituents, wherein X can be CH₃, a halogen, CF₃, OH, NH₂, OR, N₃, orCH₂X. Halogens can be F, Cl, Br, or I. R is selected from the groupconsisting of alkyl, alkene, alkyne, and aryl. Heavy oxygen 18 isdemonstrated in red.

FIG. 8 provides examples of specific heavy oxygen 18 labeled thymidinesof the invention. Some are singly labeled and some comprise two or more¹⁸Os. Heavy oxygen 18 is demonstrated in red.

FIG. 9 provides examples of specific heavy oxygen labeled 2′-deoxyuridinethymidines (also referred to as 2′-deoxyuridine) of theinvention. Some are singly labeled and others comprise two or more ¹⁸Os.Heavy oxygen 18 is demonstrated in red.

DETAILED DESCRIPTION

Abbreviations and Acronyms

CT—computerized tomography (also referred to as computerized axialtomography or CAT)

FDG—fluorodeoxyglucose

Gy—gray, the international system (SI) unit of radiation dose, expressedas absorbed energy per unit mass of tissue; Gy has replaced “rad”

GyE—gray equivalent

HT—heavy thymidine, also referred to as HTd

¹⁸O—oxygen-18

PAAM—proton activated atomic medicine

PBT—proton beam therapy

PET—positron emission tomography

SOBP—spread-out bragg peak

Td—thymidine

Definitions

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below. Unlessdefined otherwise, all technical and scientific terms used herein havethe commonly understood meaning by one of ordinary skill in the art towhich the invention pertains. Although any methods and materials similaror equivalent to those described herein may be useful in the practice ortesting of the present invention, preferred methods and materials aredescribed below. Specific terminology of particular importance to thedescription of the present invention is defined below.

2′-deoxyuridine is also referred to as (2-Deoxy-β-D-ribofuranosyl)uraciland uracil deoxyriboside.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “about,” as used herein, means approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 10%. In one aspect, the term “about” meansplus or minus 10% of the numerical value of the number with which it isbeing used. Therefore, about 50% means in the range of 45%-55%.Numerical ranges recited herein by endpoints include all numbers andfractions subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbersand fractions thereof are presumed to be modified by the term “about.”

The terms “additional therapeutically active compound” or “additionaltherapeutic agent”, as used in the context of the present invention,refers to the use or administration of a compound for an additionaltherapeutic use for a particular injury, disease, or disorder beingtreated. Such a compound, for example, could include one being used totreat an unrelated disease or disorder, or a disease or disorder whichmay not be responsive to the primary treatment for the injury, diseaseor disorder being treated.

As used herein, the term “adjuvant” refers to a substance that elicitsan enhanced immune response when used in combination with a specificantigen.

As use herein, the terms “administration of” and or “administering” acompound should be understood to mean providing a compound of theinvention or a prodrug of a compound of the invention to a subject inneed of treatment.

The term “adult” as used herein, is meant to refer to any non-embryonicor non-juvenile subject. For example the term “adult adipose tissue stemcell,” refers to an adipose stem cell, other than that obtained from anembryo or juvenile subject.

As used herein, an “agonist” is a composition of matter which, whenadministered to a mammal such as a human, enhances or extends abiological activity attributable to the level or presence of a targetcompound or molecule of interest in the mammal.

A disease or disorder is “alleviated” if the severity of a symptom ofthe disease, condition, or disorder, or the frequency with which such asymptom is experienced by a subject, or both, are reduced.

As used herein, “alleviating a disease or disorder symptom,” meansreducing the severity of the symptom or the frequency with which such asymptom is experienced by a subject, or both.

An “antagonist” is a composition of matter which when administered to amammal such as a human, inhibits a biological activity attributable tothe level or presence of a compound or molecule of interest in themammal.

As used herein, amino acids are represented by the full name thereof, bythe three letter code corresponding thereto, or by the one-letter codecorresponding thereto, as indicated in the following table:

Full Name Three-Letter Code One-Letter Code Aspartic Acid Asp D GlutamicAcid Glu E Lysine Lys K Arginine Arg R Histidine His H Tyrosine Tyr YCysteine Cys C Asparagine Asn N Glutamine Gln Q Serine Ser S ThreonineThr T Glycine Gly G Alanine Ala A Valine Val V Leucine Leu L IsoleucineIle I Methionine Met M Proline Pro P Phenylalanine Phe F Tryptophan TrpW

The expression “amino acid” as used herein is meant to include bothnatural and synthetic amino acids, and both D and L amino acids.“Standard amino acid” means any of the twenty standard L-amino acidscommonly found in naturally occurring peptides. “Nonstandard amino acidresidue” means any amino acid, other than the standard amino acids,regardless of whether it is prepared synthetically or derived from anatural source. As used herein, “synthetic amino acid” also encompasseschemically modified amino acids, including but not limited to salts,amino acid derivatives (such as amides), and substitutions. Amino acidscontained within the peptides of the present invention, and particularlyat the carboxy- or amino-terminus, can be modified by methylation,amidation, acetylation or substitution with other chemical groups whichcan change the peptide's circulating half-life without adverselyaffecting their activity. Additionally, a disulfide linkage may bepresent or absent in the peptides of the invention.

The term “amino acid” is used interchangeably with “amino acid residue,”and may refer to a free amino acid and to an amino acid residue of apeptide. It will be apparent from the context in which the term is usedwhether it refers to a free amino acid or a residue of a peptide.

Amino acids have the following general structure:

Amino acids may be classified into seven groups on the basis of the sidechain R: (1) aliphatic side chains, (2) side chains containing ahydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) sidechains containing an acidic or amide group, (5) side chains containing abasic group, (6) side chains containing an aromatic ring, and (7)proline, an imino acid in which the side chain is fused to the aminogroup.

The nomenclature used to describe the peptide compounds of the presentinvention follows the conventional practice wherein the amino group ispresented to the left and the carboxy group to the right of each aminoacid residue. In the formulae representing selected specific embodimentsof the present invention, the amino-and carboxy-terminal groups,although not specifically shown, will be understood to be in the formthey would assume at physiologic pH values, unless otherwise specified.

The term “basic” or “positively charged” amino acid as used herein,refers to amino acids in which the R groups have a net positive chargeat pH 7.0, and include, but are not limited to, the standard amino acidslysine, arginine, and histidine.

As used herein, an “analog”, or “analogue”, of a chemical compound is acompound that, by way of example, resembles another in structure but isnot necessarily an isomer (e.g., 5-fluorouracil is an analog ofthymine).

An “antagonist” is a composition of matter which when administered to amammal such as a human, inhibits a biological activity attributable tothe level or presence of a compound or molecule of interest in thesubject.

The term “antibody”, as used herein, refers to an immunoglobulinmolecule which is able to specifically bind to a specific epitope on anantigen. Antibodies can be intact immunoglobulins derived from naturalsources or from recombinant sources and can be immunoreactive portionsof intact immunoglobulins. Antibodies are typically tetramers ofimmunoglobulin molecules. The antibodies in the present invention mayexist in a variety of forms including, for example, polyclonalantibodies, monoclonal antibodies, Fv, Fab and F(ab)₂, as well as singlechain antibodies and humanized antibodies. An “antibody heavy chain”, asused herein, refers to the larger of the two types of polypeptide chainspresent in all antibody molecules.

An “antibody light chain”, as used herein, refers to the smaller of thetwo types of polypeptide chains present in all antibody molecules.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

The term “antigen” as used herein is defined as a molecule that provokesan immune response. This immune response may involve either antibodyproduction, or the activation of specific immunologically-competentcells, or both. An antigen can be derived from organisms, subunits ofproteins/antigens, killed or inactivated whole cells or lysates.

The term “antigenic determinant” as used herein refers to that portionof an antigen that makes contact with a particular antibody (i.e., anepitope). When a protein or fragment of a protein, or chemical moiety isused to immunize a host animal, numerous regions of the antigen mayinduce the production of antibodies that bind specifically to a givenregion or three-dimensional structure on the protein; these regions orstructures are referred to as antigenic determinants. An antigenicdeterminant may compete with the intact antigen (i.e., the “immunogen”used to elicit the immune response) for binding to an antibody.

The term “antimicrobial agents” as used herein refers to anynaturally-occurring, synthetic, or semi-synthetic compound orcomposition or mixture thereof, which is safe for human or animal use aspracticed in the methods of this invention, and is effective in killingor substantially inhibiting the growth of microbes. “Antimicrobial” asused herein, includes antibacterial, antifungal, and antiviral agents.

“Anti-proliferative,” as used herein, refers to the ability of acompound to impede or inhibit cell proliferation. As such, the compoundmay act directly on a cell or may act indirectly. For example, in thecontext of cancer, a cancer cell can be inhibited from proliferating bydepriving it of blood supply. The term “anti-proliferative” does notrefer to a particular mechanism by which proliferation is inhibited orimpeded.

As used herein the term “anti-tumor agent” relates to agents known inthe art that have been demonstrated to have utility for treatingneoplastic disease. For example, antitumor agents include, but are notlimited to, antibodies, toxins, chemotherapeutics, enzymes, cytokines,radionuclides, photodynamic agents, and angiogenesis inhibitors. Toxinsinclude ricin A chain, mutant Pseudomonas exotoxins, diphtheria toxoid,streptonigrin, boamycin, saporin, gelonin, and pokeweed antiviralprotein. Chemotherapeutics include 5-fluorouracil (5-FU), daunorubicin,cisplatinum, bleomycin, melphalan, taxol, tamoxifen, mitomycin-C, andmethotrexate as well as any of the compounds described in U.S. Pat. No.6,372,719 (the disclosure of which is incorporated herein by reference)as being chemotherapeutic agents. Radionuclides include radiometals.Photodynamic agents include porphyrins and their derivatives.

As used herein, the term “antisense oligonucleotide” or antisensenucleic acid means a nucleic acid polymer, at least a portion of whichis complementary to a nucleic acid which is present in a normal cell orin an affected cell. “Antisense” refers particularly to the nucleic acidsequence of the non-coding strand of a double stranded DNA moleculeencoding a protein, or to a sequence which is substantially homologousto the non-coding strand. As defined herein, an antisense sequence iscomplementary to the sequence of a double stranded DNA molecule encodinga protein. It is not necessary that the antisense sequence becomplementary solely to the coding portion of the coding strand of theDNA molecule. The antisense sequence may be complementary to regulatorysequences specified on the coding strand of a DNA molecule encoding aprotein, which regulatory sequences control expression of the codingsequences. The antisense oligonucleotides of the invention include, butare not limited to, phosphorothioate oligonucleotides and othermodifications of oligonucleotides.

The term “binding” refers to the adherence of molecules to one another,such as, but not limited to, enzymes to substrates, ligands toreceptors, antibodies to antigens, DNA binding domains of proteins toDNA, and DNA or RNA strands to complementary strands.

“Binding partner,” as used herein, refers to a molecule capable ofbinding to another molecule.

The term “biocompatible”, as used herein, refers to a material that doesnot elicit a substantial detrimental response in the host.

As used herein, the term “biologically active fragments” or “bioactivefragment” of the polypeptides encompasses natural or synthetic portionsof the full-length protein that are capable of specific binding to theirnatural ligand or of performing the function of the protein.

The term “biological sample,” as used herein, refers to samples obtainedfrom a subject, including, but not limited to, sputum, mucus, phlegm,tissues, biopsies, cerebrospinal fluid, blood, serum, plasma, otherblood components, gastric aspirates, throat swabs, pleural effusion,peritoneal fluid, follicular fluid, ascites, skin, hair, tissue, blood,plasma, cells, saliva, sweat, tears, semen, stools, Pap smears, andurine. One of skill in the art will understand the type of sampleneeded.

A “biomarker” or “marker” is a specific biochemical in the body whichhas a particular molecular feature that makes it useful for measuringthe progress of disease or the effects of treatment, or for measuring aprocess of interest. The term “cancer”, as used herein, is defined asproliferation of cells whose unique trait—loss of normalcontrols—results in unregulated growth, lack of differentiation, localtissue invasion, and metastasis. Examples include, but are not limitedto, carcinomas, sarcomas, leukemias, glioblastoma, melanoma, breastcancer, prostate cancer, ovarian cancer, uterine cancer, cervicalcancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer,and lung cancer. “Cancer” or “malignancy” are used as synonymous termsand refer to any of a number of diseases that are characterized byuncontrolled, abnormal proliferation of cells, the ability of affectedcells to spread locally or through the bloodstream and lymphatic systemto other parts of the body (i.e., metastasize), as well as any of anumber of characteristic structural and/or molecular features. A“cancerous” or “malignant cell” is understood as a cell having specificstructural properties, lacking differentiation and being capable ofinvasion and metastasis. Examples of cancers are, breast, lung, brain,bone, liver, kidney, colon, and prostate cancer. (see DeVita, V. et al.(eds.), 2001, Cancer Principles and Practice of Oncology, 6th. Ed.,Lippincott Williams & Wilkins, Philadelphia, Pa.; this reference isherein incorporated by reference in its entirety for all purposes).

“Cancer-associated” refers to the relationship of a nucleic acid and itsexpression, or lack thereof, or a protein and its level or activity, orlack thereof, to the onset of malignancy in a subject cell. For example,cancer can be associated with expression of a particular gene that isnot expressed, or is expressed at a lower level, in a normal healthycell. Conversely, a cancer-associated gene can be one that is notexpressed in a malignant cell (or in a cell undergoing transformation),or is expressed at a lower level in the malignant cell than it isexpressed in a normal healthy cell.

As used herein, the term “carrier molecule” refers to any molecule thatis chemically conjugated to the antigen of interest that enables animmune response resulting in antibodies specific to the native antigen.

As used herein, the term “chemically conjugated,” or “conjugatingchemically” refers to linking the antigen to the carrier molecule. Thislinking can occur on the genetic level using recombinant technology,wherein a hybrid protein may be produced containing the amino acidsequences, or portions thereof, of both the antigen and the carriermolecule. This hybrid protein is produced by an oligonucleotide sequenceencoding both the antigen and the carrier molecule, or portions thereof.This linking also includes covalent bonds created between the antigenand the carrier protein using other chemical reactions, such as, but notlimited to glutaraldehyde reactions. Covalent bonds may also be createdusing a third molecule bridging the antigen to the carrier molecule.These cross-linkers are able to react with groups, such as but notlimited to, primary amines, sulfhydryls, carbonyls, carbohydrates, orcarboxylic acids, on the antigen and the carrier molecule. Chemicalconjugation also includes non-covalent linkage between the antigen andthe carrier molecule.

The term “competitive sequence” refers to a peptide or a modification,fragment, derivative, or homolog thereof that competes with anotherpeptide for its cognate binding site.

“Complementary” refers to the broad concept of sequence complementaritybetween regions of two nucleic acid strands or between two regions ofthe same nucleic acid strand. It is known that an adenine residue of afirst nucleic acid region is capable of forming specific hydrogen bonds(“base pairing”) with a residue of a second nucleic acid region which isantiparallel to the first region if the residue is thymine or uracil. Asused herein, the terms “complementary” or “complementarity” are used inreference to polynucleotides (i.e., a sequence of nucleotides) relatedby the base-pairing rules. For example, for the sequence “A-G-T,” iscomplementary to the sequence “T-C-A.”

Similarly, it is known that a cytosine residue of a first nucleic acidstrand is capable of base pairing with a residue of a second nucleicacid strand which is antiparallel to the first strand if the residue isguanine. A first region of a nucleic acid is complementary to a secondregion of the same or a different nucleic acid if, when the two regionsare arranged in an antiparallel fashion, at least one nucleotide residueof the first region is capable of base pairing with a residue of thesecond region. Preferably, the first region comprises a first portionand the second region comprises a second portion, whereby, when thefirst and second portions are arranged in an antiparallel fashion, atleast about 50%, and preferably at least about 75%, at least about 90%,or at least about 95% of the nucleotide residues of the first portionare capable of base pairing with nucleotide residues in the secondportion. More preferably, all nucleotide residues of the first portionare capable of base pairing with nucleotide residues in the secondportion.

The term “complex”, as used herein in reference to proteins, refers tobinding or interaction of two or more proteins. Complex formation orinteraction can include such things as binding, changes in tertiarystructure, and modification of one protein by another, such asphosphorylation.

A “compound”, as used herein, refers to any type of substance or agentthat is commonly considered a drug, or a candidate for use as a drug, aswell as combinations and mixtures of the above. When referring to acompound of the invention, and unless otherwise specified, the term“compound” is intended to encompass not only the specified molecularentity but also its pharmaceutically acceptable, pharmacologicallyactive analogs, including, but not limited to, salts, polymorphs,esters, amides, prodrugs, adducts, conjugates, active metabolites, andthe like, where such modifications to the molecular entity areappropriate.

A “computer-readable medium” is an information storage medium that canbe accessed by a computer using a commercially available or custom-madeinterface. Exemplary computer-readable media include memory (e.g., RAM,ROM, flash memory, etc.), optical storage media (e.g., CD-ROM), magneticstorage media (e.g., computer hard drives, floppy disks, etc.), punchcards, or other commercially available media. Information may betransferred between a system of interest and a medium, betweencomputers, or between computers and the computer-readable medium forstorage or access of stored information. Such transmission can beelectrical, or by other available methods, such as IR links, wirelessconnections, etc.

As used herein, the term “conservative amino acid substitution” isdefined herein as an amino acid exchange within one of the followingfive groups:

I. Small aliphatic, nonpolar or slightly polar residues:

-   -   Ala, Ser, Thr, Pro, Gly;

II. Polar, negatively charged residues and their amides:

-   -   Asp, Asn, Glu, Gln;

III. Polar, positively charged residues:

-   -   His, Arg, Lys;

IV. Large, aliphatic, nonpolar residues:

-   -   Met Leu, Ile, Val, Cys

V. Large, aromatic residues:

-   -   Phe, Tyr, Trp

A “control” cell is a cell having the same cell type as a test cell. Thecontrol cell may, for example, be examined at precisely or nearly thesame time the test cell is examined. The control cell may also, forexample, be examined at a time distant from the time at which the testcell is examined, and the results of the examination of the control cellmay be recorded so that the recorded results may be compared withresults obtained by examination of a test cell.

A “test” cell is a cell being examined.

A “pathoindicative” cell is a cell which, when present in a tissue, isan indication that the animal in which the tissue is located (or fromwhich the tissue was obtained) is afflicted with a disease or disorder.

A “pathogenic” cell is a cell which, when present in a tissue, causes orcontributes to a disease or disorder in the animal in which the tissueis located (or from which the tissue was obtained).

A tissue “normally comprises” a cell if one or more of the cell arepresent in the tissue in an animal not afflicted with a disease ordisorder.

The term “delivery vehicle” refers to any kind of device or materialwhich can be used to deliver compounds in vivo or can be added to acomposition comprising compounds administered to a plant or animal. Thisincludes, but is not limited to, implantable devices, aggregates ofcells, matrix materials, gels, etc.

As used herein, a “derivative” of a compound refers to a chemicalcompound that may be produced from another compound of similar structurein one or more steps, as in replacement of H by an alkyl, acyl, or aminogroup.

The use of the word “detect” and its grammatical variants refers tomeasurement of the species without quantification, whereas use of theword “determine” or “measure” with their grammatical variants are meantto refer to measurement of the species with quantification. The terms“detect” and “identify” are used interchangeably herein.

As used herein, a “detectable marker” or a “reporter molecule” is anatom or a molecule that permits the specific detection of a compoundcomprising the marker in the presence of similar compounds without amarker. Detectable markers or reporter molecules include, e.g.,radioactive isotopes, antigenic determinants, enzymes, nucleic acidsavailable for hybridization, chromophores, fluorophores,chemiluminescent molecules, electrochemically detectable molecules, andmolecules that provide for altered fluorescence-polarization or alteredlight-scattering.

As used herein, the term “diagnosis” refers to detecting cancer or arisk or propensity for development of cancer, for the types of cancerencompassed by the invention. In any method of diagnosis exist falsepositives and false negatives. Any one method of diagnosis does notprovide 100% accuracy.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate.

In contrast, a “disorder” in an animal is a state of health in which theanimal is able to maintain homeostasis, but in which the animal's stateof health is less favorable than it would be in the absence of thedisorder. Left untreated, a disorder does not necessarily cause afurther decrease in the animal's state of health.

As used herein, the term “domain” refers to a part of a molecule orstructure that shares common physicochemical features, such as, but notlimited to, hydrophobic, polar, globular and helical domains orproperties such as ligand binding, signal transduction, cell penetrationand the like. Specific examples of binding domains include, but are notlimited to, DNA binding domains and ATP binding domains.

As used herein, an “effective amount” or “therapeutically effectiveamount” means an amount sufficient to produce a selected effect, such asalleviating symptoms of a disease or disorder. In the context ofadministering compounds in the form of a combination, such as multiplecompounds, the amount of each compound, when administered in combinationwith another compound(s), may be different from when that compound isadministered alone. Thus, an effective amount of a combination ofcompounds refers collectively to the combination as a whole, althoughthe actual amounts of each compound may vary. The term “more effective”means that the selected effect is alleviated to a greater extent by onetreatment relative to the second treatment to which it is beingcompared.

“Element” refers to a species of atoms; all atoms with the same numberof protons in the atomic nucleus- a pure chemical substance composed ofatoms with the same number of protons in the atomic nucleus

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

As used herein, the term “effector domain” refers to a domain capable ofdirectly interacting with an effector molecule, chemical, or structurein the cytoplasm which is capable of regulating a biochemical pathway.

As used in the specification and the appended claims, the terms “forexample”, “for instance,” “such as”, “including” and the like are meantto introduce examples that further clarify more general subject matter.Unless otherwise specified, these examples are provided only as an aidfor understanding the invention, and are not meant to be limiting in anyfashion.

The terms “formula” and “structure” are used interchangeably herein. An“enhancer” is a DNA regulatory element that can increase the efficiencyof transcription, regardless of the distance or orientation of theenhancer relative to the start site of transcription.

The term “epitope” as used herein is defined as small chemical groups onthe antigen molecule that can elicit and react with an antibody. Anantigen can have one or more epitopes. Most antigens have many epitopes;i.e., they are multivalent. In general, an epitope is roughly 5 aminoacids or sugars in size. One skilled in the art understands thatgenerally the overall three-dimensional structure, rather than thespecific linear sequence of the molecule, is the main criterion ofantigenic specificity.

As used herein, an “essentially pure” preparation of a particularprotein or peptide is a preparation wherein at least about 95%, andpreferably at least about 99%, by weight, of the protein or peptide inthe preparation is the particular protein or peptide.

A “fragment” or “segment” is a portion of an amino acid sequence,comprising at least one amino acid, or a portion of a nucleic acidsequence comprising at least one nucleotide. The terms “fragment” and“segment” are used interchangeably herein.

As used herein, a “functional” molecule is a molecule in a form in whichit exhibits a property or activity by which it is characterized. Afunctional enzyme, for example, is one that exhibits the characteristiccatalytic activity by which the enzyme is characterized.

“Gamma rays” (gamma irradiation) refers to a stream of high-energyelectromagnetic radiation given off by an atomic nucleus undergoingradioactive decay. The energies of gamma rays are higher than those ofX-rays; thus, gamma rays have greater penetrating power.

“Half-life” (radioactive) refers to the time interval that it takes forthe total number of atoms of any radioactive isotope to decay and leaveonly one-half of the original number of atoms.

“Homologous” as used herein, refers to the subunit sequence similaritybetween two polymeric molecules, e.g., between two nucleic acidmolecules, e.g., two DNA molecules or two RNA molecules, or between twopolypeptide molecules.

When a subunit position in both of the two molecules is occupied by thesame monomeric subunit, e.g., if a position in each of two DNA moleculesis occupied by adenine, then they are homologous at that position. Thehomology between two sequences is a direct function of the number ofmatching or homologous positions, e.g., if half (e.g., five positions ina polymer ten subunits in length) of the positions in two compoundsequences are homologous then the two sequences are 50% homologous, if90% of the positions, e.g., 9 of 10, are matched or homologous, the twosequences share 90% homology. By way of example, the DNA sequences3′ATTGCCS' and 3′TATGGC share 50% homology.

As used herein, “homology” is used synonymously with “identity.”

The determination of percent identity between two nucleotide or aminoacid sequences can be accomplished using a mathematical algorithm. Forexample, a mathematical algorithm useful for comparing two sequences isthe algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA87:2264-2268), modified as in Karlin and Altschul (1993, Proc. Natl.Acad. Sci. USA 90:5873-5877). This algorithm is incorporated into theNBLAST and XBLAST programs of Altschul, et al. (1990, J. Mol. Biol.215:403-410), and can be accessed, for example at the National Centerfor Biotechnology Information (NCBI) world wide web site. BLASTnucleotide searches can be performed with the NBLAST program (designated“blastn” at the NCBI web site), using the following parameters: gappenalty=5; gap extension penalty=2; mismatch penalty=3; match reward=1;expectation value 10.0; and word size=11 to obtain nucleotide sequenceshomologous to a nucleic acid described herein. BLAST protein searchescan be performed with the XBLAST program (designated “blastn” at theNCBI web site) or the NCBI “blastp” program, using the followingparameters: expectation value 10.0, BLOSUM62 scoring matrix to obtainamino acid sequences homologous to a protein molecule described herein.To obtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al. (1997, Nucleic Acids Res.25:3389-3402). Alternatively, PSI-Blast or PHI-Blast can be used toperform an iterated search which detects distant relationships betweenmolecules (Id.) and relationships between molecules which share a commonpattern. When utilizing BLAST, Gapped BLAST, PSI-Blast, and PHI-Blastprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically exact matches arecounted.

The term “inhibit,” as used herein, refers to the ability of a compound,agent, or method to reduce or impede a described function, level,activity, rate, etc., based on the context in which the term “inhibit”is used. Preferably, inhibition is by at least 10%, more preferably byat least 25%, even more preferably by at least 50%, and most preferably,the function is inhibited by at least 75%. The term “inhibit” is usedinterchangeably with “reduce” and “block.”

The term “inhibit a complex,” as used herein, refers to inhibiting theformation of a complex or interaction of two or more proteins, as wellas inhibiting the function or activity of the complex. The term alsoencompasses disrupting a formed complex. However, the term does notimply that each and every one of these functions must be inhibited atthe same time.

The term “inhibit a protein,” as used herein, refers to any method ortechnique which inhibits protein synthesis, levels, activity, orfunction, as well as methods of inhibiting the induction or stimulationof synthesis, levels, activity, or function of the protein of interest.The term also refers to any metabolic or regulatory pathway which canregulate the synthesis, levels, activity, or function of the protein ofinterest. The term includes binding with other molecules and complexformation. Therefore, the term “protein inhibitor” refers to any agentor compound, the application of which results in the inhibition ofprotein function or protein pathway function. However, the term does notimply that each and every one of these functions must be inhibited atthe same time.

As used herein “injecting or applying” includes administration of acompound of the invention by any number of routes and means including,but not limited to, topical, oral, buccal, intravenous, intramuscular,intra-arterial, intramedullary, intrathecal, intraventricular,transdermal, subcutaneous, intraperitoneal, intranasal, enteral,topical, sublingual, vaginal, ophthalmic, pulmonary, or rectal means.Compounds or agents of the invention can be administered to a subject bythese means when appropriate.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the peptide of the invention inthe kit for effecting alleviation of the various diseases or disordersrecited herein. Optionally, or alternately, the instructional materialmay describe one or more methods of alleviating the diseases ordisorders in a cell or a tissue of a mammal. The instructional materialof the kit of the invention may, for example, be affixed to a containerwhich contains the identified compound invention or be shipped togetherwith a container which contains the identified compound. Alternatively,the instructional material may be shipped separately from the containerwith the intention that the instructional material and the compound beused cooperatively by the recipient.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, e.g., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, e.g., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, e.g., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

“Isotope” refers to one of two or more species of atoms of a givenelement (having the same number of protons in the nucleus) withdifferent atomic masses (different number of neutrons in the nucleus).The atom can either be a stable isotope or a radioactive isotope.

“Isotopically labeled” refers to a mixture of an isotopically unmodifiedcompound with one or more analogous isotopically substitutedcompound(s).

A “ligand” is a compound that specifically binds to a target receptor ortarget molecule.

A “receptor” or target molecule is a compound that specifically binds toa ligand.

A ligand or a receptor “specifically binds to” a compound when theligand or receptor functions in a binding reaction which isdeterminative of the presence of the compound in a sample ofheterogeneous compounds. Thus, under designated assay (e.g.,immunoassay) conditions, the ligand or receptor binds preferentially toa particular compound and does not bind in a significant amount to othercompounds present in the sample. For example, a polynucleotidespecifically binds under hybridization conditions to a compoundpolynucleotide comprising a complementary sequence; an antibodyspecifically binds under immunoassay conditions to an antigen bearing anepitope against which the antibody was raised.

As used herein, the term “linkage” refers to a connection between twogroups. The connection can be either covalent or non-covalent, includingbut not limited to ionic bonds, hydrogen bonding, andhydrophobic/hydrophilic interactions.

As used herein, the term “linker” refers to a molecule that joins twoother molecules either covalently or noncovalently, e.g., through ionicor hydrogen bonds or van der Waals interactions, e.g., a nucleic acidmolecule that hybridizes to one complementary sequence at the 5′ end andto another complementary sequence at the 3′ end, thus joining twonon-complementary sequences.

“Malexpression” of a gene means expression of a gene in a cell of apatient afflicted with a disease or disorder, wherein the level ofexpression (including non-expression), the portion of the geneexpressed, or the timing of the expression of the gene with regard tothe cell cycle, differs from expression of the same gene in a cell of apatient not afflicted with the disease or disorder. It is understoodthat malexpression may cause or contribute to the disease or disorder,be a symptom of the disease or disorder, or both.

The term “measuring the level of expression” or “determining the levelof expression” as used herein refers to any measure or assay which canbe used to correlate the results of the assay with the level ofexpression of a gene, mRNA, miRNA, SNPs, or protein of interest. Suchassays include measuring the level of mRNA, protein levels, etc. and canbe performed by assays such as northern and western blot analyses,binding assays, immunoblots, etc. The level of expression can includerates of expression and can be measured in terms of the actual amount ofan mRNA or protein present. Such assays are coupled with processes orsystems to store and process information and to help quantify levels,signals, etc. and to digitize the information for use in comparinglevels.

What is meant by a “method of treating a tumor using proton beam therapyin a subject in need thereof” is meant treating cancers that aresusceptible to using proton beam therapy based on their size, location,etc.

As used herein, the term “nucleic acid” encompasses RNA as well assingle and double-stranded DNA and cDNA. Furthermore, the terms,“nucleic acid,” “DNA,” “RNA” and similar terms also include nucleic acidanalogs, i.e. analogs having other than a phosphodiester backbone. Forexample, the so-called “peptide nucleic acids,” which are known in theart and have peptide bonds instead of phosphodiester bonds in thebackbone, are considered within the scope of the present invention. By“nucleic acid” is meant any nucleic acid, whether composed ofdeoxyribonucleosides or ribonucleosides, and whether composed ofphosphodiester linkages or modified linkages such as phosphotriester,phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate,carbamate, thioether, bridged phosphoramidate, bridged methylenephosphonate, bridged phosphoramidate, bridged phosphoramidate, bridgedmethylene phosphonate, phosphorothioate, methylphosphonate,phosphorodithioate, bridged phosphorothioate or sulfone linkages, andcombinations of such linkages. The term nucleic acid also specificallyincludes nucleic acids composed of bases other than the fivebiologically occurring bases (adenine, guanine, thymine, cytosine anduracil). Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end; the left-hand direction of a double-strandedpolynucleotide sequence is referred to as the 5′-direction. Thedirection of 5′ to 3′ addition of nucleotides to nascent RNA transcriptsis referred to as the transcription direction. The DNA strand having thesame sequence as an mRNA is referred to as the “coding strand”;sequences on the DNA strand which are located 5′ to a reference point onthe DNA are referred to as “upstream sequences”; sequences on the DNAstrand which are 3′ to a reference point on the DNA are referred to as“downstream sequences.”

The term “nucleic acid construct,” as used herein, encompasses DNA andRNA sequences encoding the particular gene or gene fragment desired,whether obtained by genomic or synthetic methods.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

The term “Oligonucleotide” typically refers to short polynucleotides,generally no greater than about 50 nucleotides. It will be understoodthat when a nucleotide sequence is represented by a DNA sequence (i.e.,A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) inwhich “U” replaces “T.”

“Operably linked” refers to a juxtaposition wherein the components areconfigured so as to perform their usual function. Thus, controlsequences or promoters operably linked to a coding sequence are capableof effecting the expression of the coding sequence. By describing twopolynucleotides as “operably linked” is meant that a single-stranded ordouble-stranded nucleic acid moiety comprises the two polynucleotidesarranged within the nucleic acid moiety in such a manner that at leastone of the two polynucleotides is able to exert a physiological effectby which it is characterized upon the other. By way of example, apromoter operably linked to the coding region of a gene is able topromote transcription of the coding region.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intraperitoneal, intramuscular, intrasternal injection, and kidneydialytic infusion techniques.

The term “peptide” typically refers to short polypeptides.

As used herein, the term “peptide ligand” (or the word “ligand” inreference to a peptide) refers to a peptide or fragment of a proteinthat specifically binds to a molecule, such as a protein, carbohydrate,and the like. A receptor or binding partner of the peptide ligand can beessentially any type of molecule such as polypeptide, nucleic acid,carbohydrate, lipid, or any organic derived compound. Specific examplesof ligands are peptide ligands of the present inventions.

The term “per application” as used herein refers to administration of adrug or compound to a subject.

The term “pharmaceutical composition” shall mean a compositioncomprising at least one active ingredient, whereby the composition isamenable to investigation for a specified, efficacious outcome in amammal (for example, without limitation, a human). Those of ordinaryskill in the art will understand and appreciate the techniquesappropriate for determining whether an active ingredient has a desiredefficacious outcome based upon the needs of the artisan.

As used herein, the term “pharmaceutically-acceptable carrier” means achemical composition with which an appropriate compound or derivativecan be combined and which, following the combination, can be used toadminister the appropriate compound to a subject.

As used herein, the term “physiologically acceptable” ester or saltmeans an ester or salt form of the active ingredient which is compatiblewith any other ingredients of the pharmaceutical composition, which isnot deleterious to the subject to which the composition is to beadministered.

“Pharmaceutically acceptable” means physiologically tolerable, foreither human or veterinary application.

As used herein, “pharmaceutical compositions” include formulations forhuman and veterinary use.

“Plurality” means at least two.

“Pharmaceutically acceptable” means physiologically tolerable, foreither human or veterinary application.

As used herein, “pharmaceutical compositions” include formulations forhuman and veterinary use.

“Plurality” means at least two.

“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof.

“Synthetic peptides or polypeptides” means a non-naturally occurringpeptide or polypeptide. Synthetic peptides or polypeptides can besynthesized, for example, using an automated polypeptide synthesizer.Various solid phase peptide synthesis methods are known to those ofskill in the art. “Positron” refers to the antimatter counterpart of theelectron, with a mass identical to that of the electron and an equal butopposite (positive) charge.

“Positron emission tomography (PET) scan” refers to an imaging techniquethat is used to observe metabolic activity within the body. The systemdetects pairs of gamma rays emitted indirectly by a radioactive isotopeused as a tracer, which emits positrons and which is introduced into thebody on a biologically-active molecule. Three-dimensional images of theconcentration of the radioactive isotope within the body are thenconstructed by computer analysis. The imaging often is performed with anX-ray CT scan in the same instrument.

“Proton” refers to an elementary particle having a rest mass of about1.673×10-27 kg, slightly less than that of a neutron, and a positiveelectric charge equal and opposite to that of the electron. The numberof protons in the nucleus of an atom is the atomic number.

As used herein, the term “providing a prognosis” refers to providinginformation regarding the impact of the presence of cancer (e.g., asdetermined by the diagnostic methods of the present invention) on asubject's future health (e.g., expected morbidity or mortality, thelikelihood of getting cancer, and the risk of metastasis).

The term “prevent,” as used herein, means to stop something fromhappening, or taking advance measures against something possible orprobable from happening. In the context of medicine, “prevention”generally refers to action taken to decrease the chance of getting adisease or condition.

A “preventive” or “prophylactic” treatment is a treatment administeredto a subject who does not exhibit signs, or exhibits only early signs,of a disease or disorder. A prophylactic or preventative treatment isadministered for the purpose of decreasing the risk of developingpathology associated with developing the disease or disorder. Unless theterm “treatment” or “treating” is used with the term preventive orprophylactic treatment it should not be construed to include such inless it is clear by the context.

A “prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or injury or exhibits only earlysigns of the disease or injury for the purpose of decreasing the risk ofdeveloping pathology associated with the disease or injury. The term“protein regulatory pathway”, as used herein, refers to both theupstream regulatory pathway which regulates a protein, as well as thedownstream events which that protein regulates. Such regulationincludes, but is not limited to, transcription, translation, levels,activity, posttranslational modification, and function of the protein ofinterest, as well as the downstream events which the protein regulates.

The terms “protein pathway” and “protein regulatory pathway” are usedinterchangeably herein.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulator sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

A “constitutive” promoter is a promoter which drives expression of agene to which it is operably linked, in a constant manner in a cell. Byway of example, promoters which drive expression of cellularhousekeeping genes are considered to be constitutive promoters.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a living cell substantiallyonly when an inducer which corresponds to the promoter is present in thecell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a living cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter.

As used herein, “protecting group” with respect to a terminal aminogroup refers to a terminal amino group of a peptide, which terminalamino group is coupled with any of various amino-terminal protectinggroups traditionally employed in peptide synthesis. Such protectinggroups include, for example, acyl protecting groups such as formyl,acetyl, benzoyl, trifluoroacetyl, succinyl, and methoxysuccinyl;aromatic urethane protecting groups such as benzyloxycarbonyl; andaliphatic urethane protecting groups, for example, tert-butoxycarbonylor adamantyloxycarbonyl. See Gross and Mienhofer, eds., The Peptides,vol. 3, pp. 3-88 (Academic Press, New York, 1981) for suitableprotecting groups.

As used herein, “protecting group” with respect to a terminal carboxygroup refers to a terminal carboxyl group of a peptide, which terminalcarboxyl group is coupled with any of various carboxyl-terminalprotecting groups. Such protecting groups include, for example,tert-butyl, benzyl or other acceptable groups linked to the terminalcarboxyl group through an ester or ether bond.

As used herein, the term “purified” and like terms relate to anenrichment of a molecule or compound relative to other componentsnormally associated with the molecule or compound in a nativeenvironment. The term “purified” does not necessarily indicate thatcomplete purity of the particular molecule has been achieved during theprocess. A “highly purified” compound as used herein refers to acompound that is greater than 90% pure.

“Radioactive decay” refers to the process by which unstable (orradioactive) isotopes lose energy by emitting alpha particles (heliumnuclei), beta particles (positive or negative electrons), gammaradiation, neutrons or protons to reach a final stable energy state.

“Radioactive isotope (radioisotope)” refers to an atom for whichradioactive decay has been experimentally measured (also see half-life).

The term “regulate” refers to either stimulating or inhibiting afunction or activity of interest.

“Recombinant polynucleotide” refers to a polynucleotide having sequencesthat are not naturally joined together. An amplified or assembledrecombinant polynucleotide may be included in a suitable vector, and thevector can be used to transform a suitable host cell.

A recombinant polynucleotide may serve a non-coding function (e.g.,promoter, origin of replication, ribosome-binding site, etc.) as well.

A host cell that comprises a recombinant polynucleotide is referred toas a “recombinant host cell.” A gene which is expressed in a recombinanthost cell wherein the gene comprises a recombinant polynucleotide,produces a “recombinant polypeptide.”

A “recombinant polypeptide” is one which is produced upon expression ofa recombinant polynucleotide.

By “small interfering RNAs (siRNAs)” is meant, inter alia, an isolateddsRNA molecule comprised of both a sense and an anti-sense strand. Inone aspect, it is greater than 10 nucleotides in length. siRNA alsorefers to a single transcript which has both the sense and complementaryantisense sequences from the target gene, e.g., a hairpin. siRNA furtherincludes any form of dsRNA (proteolytically cleaved products of largerdsRNA, partially purified RNA, essentially pure RNA, synthetic RNA,recombinantly produced RNA) as well as altered RNA that differs fromnaturally occurring RNA by the addition, deletion, substitution, and/oralteration of one or more nucleotides.

As used herein, the term “solid support” relates to a solvent insolublesubstrate that is capable of forming linkages (preferably covalentbonds) with various compounds. The support can be either biological innature, such as, without limitation, a cell or bacteriophage particle,or synthetic, such as, without limitation, an acrylamide derivative,agarose, cellulose, nylon, silica, or magnetized particles.

By the term “specifically binds to”, as used herein, is meant when acompound or ligand functions in a binding reaction or assay conditionswhich is determinative of the presence of the compound in a sample ofheterogeneous compounds, or it means that one molecule, such as abinding moiety, e.g., an oligonucleotide or antibody, bindspreferentially to another molecule, such as a target molecule, e.g., anucleic acid or a protein, in the presence of other molecules in asample.

The terms “specific binding” or “specifically binding” when used inreference to the interaction of a peptide (ligand) and a receptor(molecule) also refers to an interaction that is dependent upon thepresence of a particular structure (i.e., an amino sequence of a ligandor a ligand binding domain within a protein); in other words the peptidecomprises a structure allowing recognition and binding to a specificprotein structure within a binding partner rather than to molecules ingeneral. For example, if a ligand is specific for binding pocket “A,” ina reaction containing labeled peptide ligand “A” (such as an isolatedphage displayed peptide or isolated synthetic peptide) and unlabeled “A”in the presence of a protein comprising a binding pocket A the unlabeledpeptide ligand will reduce the amount of labeled peptide ligand bound tothe binding partner, in other words a competitive binding assay.

By the term “specifically binds to”, as used herein, is meant when acompound or ligand functions in a binding reaction or assay conditionswhich is determinative of the presence of the compound in a sample ofheterogeneous compounds.

“Stable isotope” refers to an atom for which no radioactive decay hasever been experimentally measured.

The term “standard,” as used herein, refers to something used forcomparison. For example, it can be a known standard agent or compoundwhich is administered and used for comparing results when administeringa test compound, or it can be a standard parameter or function which ismeasured to obtain a control value when measuring an effect of an agentor compound on a parameter or function. Standard can also refer to an“internal standard”, such as an agent or compound which is added atknown amounts to a sample and is useful in determining such things aspurification or recovery rates when a sample is processed or subjectedto purification or extraction procedures before a marker of interest ismeasured. Internal standards are often a purified marker of interestwhich has been labeled, such as with a radioactive isotope, allowing itto be distinguished from an endogenous marker.

A “subject” of analysis, diagnosis, or treatment is an animal. Suchanimals include mammals, preferably a human.

As used herein, a “subject in need thereof” is a patient, animal,mammal, or human, who will benefit from the method of this invention.

The term “symptom,” as used herein, refers to any morbid phenomenon ordeparture from the normal in structure, function, or sensation,experienced by the patient and indicative of disease. In contrast, a“sign” is objective evidence of disease. For example, a bloody nose is asign. It is evident to the patient, doctor, nurse and other observers.

The term “substantially pure” describes a compound, e.g., a protein orpolypeptide which has been separated from components which naturallyaccompany it. Typically, a compound is substantially pure when at least10%, more preferably at least 20%, more preferably at least 50%, morepreferably at least 60%, more preferably at least 75%, more preferablyat least 90%, and most preferably at least 99% of the total material (byvolume, by wet or dry weight, or by mole percent or mole fraction) in asample is the compound of interest. Purity can be measured by anyappropriate method, e.g., in the case of polypeptides by columnchromatography, gel electrophoresis, or HPLC analysis. A compound, e.g.,a protein, is also substantially purified when it is essentially free ofnaturally associated components or when it is separated from the nativecontaminants which accompany it in its natural state.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology for the purpose of diminishing oreliminating those signs.

A “therapeutically effective amount” of a compound is that amount ofcompound which is sufficient to provide a beneficial effect to thesubject to which the compound is administered.

The term to “treat,” as used herein, means reducing the frequency withwhich symptoms are experienced by a patient or subject or administeringan agent or compound to reduce the frequency with which symptoms areexperienced.

A “prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs of thedisease for the purpose of decreasing the risk of developing pathologyassociated with the disease. Unless the term “treatment” or “treating”is used with the term “preventive” or “prophylactic” it should not beconstrued to include such in less it is clear by the context.

By the term “x-rays” is meant electromagnetic radiation with awavelength ranging from 0.01 to 10 nanometers- shorter than those of UVrays and typically longer than those of gamma rays.

Chemical Definitions

As used herein, the term “halogen” or “halo” includes bromo, chloro,fluoro, and iodo.

The term “haloalkyl” as used herein refers to an alkyl radical bearingat least one halogen substituent, for example, chloromethyl, fluoroethylor trifluoromethyl and the like.

The term “C₁-C_(n) alkyl” wherein n is an integer, as used herein,represents a branched or linear alkyl group having from one to thespecified number of carbon atoms. Typically, C₁-C₆ alkyl groups include,but are not limited to, methyl, ethyl, n-propyl, iso-propyl, butyl,iso-butyl, sec-butyl, tert-butyl, pentyl, hexyl, and the like.

The term “C₂-C₂ alkenyl” wherein n is an integer, as used herein,represents an olefinically unsaturated branched or linear group havingfrom two to the specified number of carbon atoms and at least one doublebond. Examples of such groups include, but are not limited to,1-propenyl, 2-propenyl, 1,3-butadienyl, 1-butenyl, hexenyl, pentenyl,and the like.

The term “C₂-C₂ alkynyl” wherein n is an integer refers to anunsaturated branched or linear group having from two to the specifiednumber of carbon atoms and at least one triple bond. Examples of suchgroups include, but are not limited to, 1-propynyl, 2-propynyl,1-butynyl, 2-butynyl, 1-pentynyl, and the like.

The term “C₃-C₂ cycloalkyl” wherein n=8, represents cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

As used herein the term “aryl” refers to an optionally substituted mono-or bicyclic carbocyclic ring system having one or two aromatic ringsincluding, but not limited to, phenyl, benzyl, naphthyl,tetrahydronaphthyl, indanyl, indenyl, and the like. AOptionallysubstituted aryl@ includes aryl compounds having from zero to foursubstituents, and Asubstituted aryl@ includes aryl compounds having oneor more substituents. The term (C₅-C₈ alkyl)aryl refers to any arylgroup which is attached to the parent moiety via the alkyl group.

The term “bicyclic” represents either an unsaturated or saturated stable7- to 12-membered bridged or fused bicyclic carbon ring. The bicyclicring may be attached at any carbon atom which affords a stablestructure. The term includes, but is not limited to, naphthyl,dicyclohexyl, dicyclohexenyl, and the like.

The term “halogen” or “halo” includes bromo, chloro, fluoro, and iodo.The term “heterocyclic group” refers to an optionally substituted mono-or bicyclic carbocyclic ring system containing from one to threeheteroatoms wherein the heteroatoms are selected from the groupconsisting of oxygen, sulfur, and nitrogen.

As used herein the term “heteroaryl” refers to an optionally substitutedmono- or bicyclic carbocyclic ring system having one or two aromaticrings containing from one to three heteroatoms and includes, but is notlimited to, furyl, thienyl, pyridyl and the like.

As used herein, the term “optionally substituted” refers to from zero tofour substituents, wherein the substituents are each independentlyselected. Each of the independently selected substituents may be thesame or different than other substituents.

The compounds of the present invention contain one or more asymmetriccenters in the molecule. In accordance with the present invention astructure that does not designate the stereochemistry is to beunderstood as embracing all the various optical isomers, as well asracemic mixtures thereof.

The compounds of the present invention may exist in tautomeric forms andthe invention includes both mixtures and separate individual tautomers.For example the following structure:

is understood to represent a mixture of the structures:

The terminology used herein is for the purpose of describing theparticular versions or embodiments only, and is not intended to limitthe scope of the present invention. All publications mentioned hereinare incorporated by reference in their entirety.

The term “pharmaceutically-acceptable salt” refers to salts which retainthe biological effectiveness and properties of the compounds of thepresent invention and which are not biologically or otherwiseundesirable. In many cases, the compounds of the present invention arecapable of forming acid and/or base salts by virtue of the presence ofamino and/or carboxyl groups or groups similar thereto.

Pharmaceutically-acceptable base addition salts can be prepared frominorganic and organic bases. Salts derived from inorganic bases, includeby way of example only, sodium, potassium, lithium, ammonium, calciumand magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary and tertiary amines, such asalkyl amines, dialkyl amines, trialkyl amines, substituted alkyl amines,di(substituted alkyl) amines, tri(substituted alkyl) amines, alkenylamines, dialkenyl amines, trialkenyl amines, substituted alkenyl amines,di(substituted alkenyl) amines, tri(substituted alkenyl) amines,cycloalkyl amines, di(cycloalkyl) amines, tri(cycloalkyl) amines,substituted cycloalkyl amines, disubstituted cycloalkyl amine,trisubstituted cycloalkyl amines, cycloalkenyl amines, di(cycloalkenyl)amines, tri(cycloalkenyl) amines, substituted cycloalkenyl amines,disubstituted cycloalkenyl amine, trisubstituted cycloalkenyl amines,aryl amines, diaryl amines, triaryl amines, heteroaryl amines,diheteroaryl amines, triheteroaryl amines, heterocyclic amines,diheterocyclic amines, triheterocyclic amines, mixed di- and tri-amineswhere at least two of the substituents on the amine are different andare selected from the group consisting of alkyl, substituted alkyl,alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocyclic,and the like. Also included are amines where the two or threesubstituents, together with the amino nitrogen, form a heterocyclic orheteroaryl group. Examples of suitable amines include, by way of exampleonly, isopropylamine, trimethyl amine, diethyl amine, tri(iso-propyl)amine, tri(n-propyl) amine, ethanolamine, 2-dimethylaminoethanol,tromethamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,N-alkylglucamines, theobromine, purines, piperazine, piperidine,morpholine, N-ethylpiperidine, and the like. It should also beunderstood that other carboxylic acid derivatives would be useful in thepractice of this invention, for example, carboxylic acid amides,including carboxamides, lower alkyl carboxamides, dialkyl carboxamides,and the like.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, and the like.

In one embodiment, the invention provides a composition useful as atherapeutic for treating cancer in a subject in need thereof. In oneaspect, the cancer is selected from the group consisting of melanoma,ovarian cancer, breast cancer, head and neck cancer, lung cancer, MMMT,bladder cancer, uterine cancer, endometrial cancer, liver cancer,pancreatic cancer, esophageal cancer, stomach cancer, cervical cancer,prostate cancer, adrenal cancer, lymphoma, leukemia, salivary glandcancer, bone cancer, brain cancer, cerebellar cancer, colon cancer,rectal cancer, colorectal cancer, oronasopharyngeal cancer, NPC, kidneycancer, skin cancer, basal cell carcinoma, hard palate carcinoma,squamous cell carcinoma of the tongue, meningioma, pleomorphic adenoma,astrocytoma, chondrosarcoma, cortical adenoma, hepatocellular carcinoma,pancreatic cancer, squamous cell carcinoma, and adenocarcinoma.

In one embodiment, the treatment encompasses a combination therapy.

The present invention encompasses administering the compounds of the4invention based on the particular cancer being diagnosed and/or treated,its location in the subject, etc. In one aspect, a composition isadministered by a route selected from the group consisting ofintratumoral, parenteral, intravenous, topical, and direct. In oneaspect, the compositions and methods of the invention are useful fordetecting, identifying, diagnosing, and treating cancer.

Embodiments

Proton therapy treatments are nonsurgical, noninvasive and usuallyresult in minimal side effects when combined and delivered using PencilBeam Scanning (PBS) technology. The resulting treatment provides an evenhigher degree of precision, further minimizing the overall radiationexposure to healthy tissue.

The present application addresses the preparation of novel compounds andtheir use in proton beam therapy, diagnostics, and in theragnostics.Theragnostics is a treatment strategy that combines therapeutics withdiagnostics. Coupling all of the uses together can be helpful to aphysician in setting up a personalized medicine (precision medicine)approach for the patient. For example, once an ¹⁸O atom is activated insitu using the methods of the invention, it becomes a positron emitterand can be detected with a PET scan. There is no literature that teachesor suggests using a heavy thymidine labeled with ¹⁸O that isadministered to a subject and is then activated by a proton beam andtransmutated into ¹⁸F. That is, there is no teaching or suggestion inthe art for the technology disclosed herein that allows for allowing forboth treatment and imaging. The idea of the activation beingpreferentially happening in the patient's cancer translates into“targeted cancer detection” and may be useful, for example, as anadjunct during and after treatment to monitor the tumor.

The present compounds also have advantages over other previously usedcompounds for proton beam therapy, because, for example, the half-lifeof ubiquitous atoms studied previously to provide in vivo assessment ofradiation dose localization and dosimetry is very short; whereas thehalf-life of activated ¹⁸F is 119 minutes. This makes ¹⁸F potentiallymore useful and practical for this specialized imaging procedure in theproton clinic, as well as for therapy.

The compositions include known and new compounds useful for practicingthe methods of the invention, as well as methods for making thecompounds. Techniques for preparing analogs, derivatives, andmodifications of the generic structures of the invention are known inthe art or described herein. Some examples of diseases which may betreated according to the methods of the invention are discussed hereinor are known in the art. The present invention further provides methodsfor testing compounds of the invention. Other methods for testingcompounds discovered using the methods of the invention are eitherdescribed herein or are known in the art.

When a halogen is added to a derivative, they can be F, Cl, Br, or I.When fluorine is used, it can be present in a compound as CF, CF₂, orCF₃.

The amount of time after administration of an oxygen 18 labeled compoundof the invention and a patient being subjected to a proton beam can bedetermined by one of ordinary skill in the art, but can be, for example,from about one hour to about 96 hours. Applicants did, for example, astudy in which 96 hours was used based on a consideration of the cellcycle time in most human cancer cells in an effort to get at least mostof the cancer cells labeled with the oxygen 18 labeled compound (e.g.,incorporated into DNA). Thus, a time of exposure to the compound can bechosen to help ensure that incorporation of the heavy nucleoside intoDNA occurs over at least one cell cycle in all cells. That is, in oneaspect, the “longer the better” may be a principle to adhere to but oneof ordinary skill in the art can adjust according to various parameterssuch as the type of tumor, its aggressiveness, etc. As for the abilityto infuse thymidine for a long time, there are clinical studies wheremassive thymidine infusions (grams/meter squared) have been used safelyin cancer patients. However, it is anticipated that such high doses areNOT wise or necessary to achieve radiation sensitization with theseheavy molecules. The dose can be “physiologic” based on prior studiesand what is known in the art about administration of molecules such asthymidine and their incorporation into DNA. Because the compounds of theinvention are not radioactive, there is no safety concern aboutadministration and a radiation dose or effect.

The amount of compound of the invention to be administered to a subjectcan be determined based on the disclosures provided herein and on otherfactors. For example, when thymidine is administered in rescue of highdose methotrexate, it is administered as an initial bolus injection of1.0 g of dThd per sq m, followed by constant infusion of 8 g/sq m/dayfor 72 hr. In one aspect, thymidine can be administered in high dosessuch as 75 g/sq m/24 hours.

In cases where compounds are sufficiently basic or acidic to form acidor base salts, use of the compounds as salts may be appropriate.Examples of acceptable salts are organic acid addition salts formed withacids which form a physiological acceptable anion, for example,tosylate, methanesulfonate, acetate, citrate, malonate, tartarate,succinate, benzoate, ascorbate, a-ketoglutarate, and α-glycerophosphate.Suitable inorganic salts may also be formed, including hydrochloride,sulfate, nitrate, bicarbonate, and carbonate salts.

Acceptable salts may be obtained using standard procedures well known inthe art, for example by reacting a sufficiently basic compound such asan amine with a suitable acid affording a physiologically acceptableanion. Alkali metal (for example, sodium, potassium or lithium) oralkaline earth metal (for example calcium) salts of carboxylic acids canalso be made.

In cases where compounds are sufficiently basic or acidic to form stablenontoxic acid or base salts, administration of the compounds as saltsmay be appropriate. Examples of pharmaceutically acceptable salts areorganic acid addition salts formed with acids which form a physiologicalacceptable anion, for example, tosylate, methanesulfonate, acetate,citrate, malonate, tartarate, succinate, benzoate, ascorbate,-ketoglutarate, and -glycerophosphate. Suitable inorganic salts may alsobe formed, including hydrochloride, sulfate, nitrate, bicarbonate, andcarbonate salts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

The compounds of the formulas of the invention can be formulated aspharmaceutical compositions and administered to a mammalian host, suchas a human patient in a variety of forms adapted to the chosen route ofadministration, i.e., orally or parenterally, by intravenous,intramuscular, topical or subcutaneous routes.

Thus, the present compounds may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the patient'sdiet. For oral therapeutic administration, the active compound may becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. Such compositions and preparations shouldcontain at least 0.1% of active compound. The percentage of thecompositions and preparations may, of course, be varied and mayconveniently be between about 2 to about 60% of the weight of a givenunit dosage form. The amount of active compound in such therapeuticallyuseful compositions is such that an effective dosage level will beobtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

The compound may also be administered intravenously or intraperitoneallyby infusion or injection. Solutions of the compound or its salts can beprepared in water, optionally mixed with a nontoxic surfactant.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, triacetin, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form should be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

For topical administration, the present compounds may be applied in pureform, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the present compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Useful dosages of the compounds can be determined by comparing their invitro activity, and in vivo activity in animal models. Methods for theextrapolation of effective dosages in mice, and other animals, to humansare known to the art; for example, see U.S. Pat. No. 4,938,949.

Generally, the concentration of the compound(s) of a formula of theinvention, in a liquid composition, such as a lotion, will be from about0.1-25 wt-%, preferably from about 0.5-10 wt-%. The concentration in asemi-solid or solid composition such as a gel or a powder will be about0.1-5 wt-%, preferably about 0.5-2.5 wt-%.

The amount of the compound, or an active salt or derivative thereof,required for use in treatment will vary not only with the particularsalt selected but also with the route of administration, the nature ofthe condition being treated and the age and condition of the patient andwill be ultimately at the discretion of the attendant physician orclinician.

In general, however, a suitable dose will be in the range of from about0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kg of bodyweight per day, such as 3 to about 50 mg per kilogram body weight of therecipient per day, preferably in the range of 6 to 90 mg/kg/day, mostpreferably in the range of 15 to 60 mg/kg/day.

The compound is conveniently administered in unit dosage form; forexample, containing 5 to 1000 mg, conveniently 10 to 750 mg, mostconveniently, 50 to 500 mg of active ingredient per unit dosage form.

Ideally, the active ingredient should be administered to achieve peakplasma concentrations of the active compound of from about 0.5 to about75 μM, preferably, about 1 to 50 μM, most preferably, about 2 to about30 μM. This may be achieved, for example, by the intravenous injectionof a 0.05 to 5% solution of the active ingredient, optionally in saline,or orally administered as a bolus containing about 1-100 mg of theactive ingredient. Desirable blood levels may be maintained bycontinuous infusion to provide about 0.01-5.0 mg/kg/hr or byintermittent infusions containing about 0.4-15 mg/kg of the activeingredient(s).

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four, or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple injections or by direct or topicalapplication.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the composition of the inventionfor its designated use. The instructional material of the kit of theinvention may, for example, be affixed to a container which contains thecomposition or be shipped together with a container which contains thecomposition. Alternatively, the instructional material may be shippedseparately from the container with the intention that the instructionalmaterial and the composition be used cooperatively by the recipient.

The method of the invention includes a kit comprising at least onecompound identified in the invention and an instructional material whichdescribes administering the compound or a composition comprising thecompound to a cell or a subject. This should be construed to includeother embodiments of kits that are known to those skilled in the art,such as a kit comprising a (preferably sterile) solvent suitable fordissolving or suspending the composition of the invention prior toadministering the compound to a cell or a subject. Preferably thesubject is a human.

In accordance with the present invention, as described above or asdiscussed in the Examples below, there can be employed conventionalchemical, cellular, histochemical, biochemical, molecular biology,microbiology, and in vivo techniques which are known to those of skillin the art. Such techniques are explained fully in the literature.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention. Therefore, the examples should be construed to encompass anyand all variations which become evident as a result of the teachingprovided herein.

EXAMPLES

The present application discloses compositions and methods to allow forconvergence of the increasing availability in clinical radiotherapy ofthe proton beam, with the emergence of the concept of a “theragnosticagent” in diagnostic radiology and oncology, and with our noveltherapeutic use of molecular chemistry applications of substitutedOxygen-18 (a safe, non-radioactive isotope) into critical cellulartargets that can be transmutated in vivo by proton bombardment. Thepredominant literature on ¹⁸O deals with its precursor role in theproduction of diagnostic agents like 18F-deoxyglucose for clinical PETscanning that now can be done with mini-cyclotrons in communityhospitals. Unexpected results are provided below.

Without wishing to be bound by any particular theory, it is hypothesizedherein that an ¹⁸O targeting agent can be used to target cancer cellsand that it will be selectively activated in cancer cells by protontreatment. One of the co-inventors is on the staff of the HamptonUniversity Proton Therapy Institute which houses state of the art protontherapy equipment. The presently described studies were focused on theinsertion of a non-radioactive isotope like ¹⁸O into critical cellulartargets. Disclosed herein is the preparation and use of heavy thymidinethat is incorporated into DNA. Our team has the expertise to make HTpossibly in new innovative ways for our particular purpose, plus othernucleosides that we will test in vitro. This endeavor could open a newapproach to cancer treatment.

This design science may lead to a clinically feasible treatment entitled“proton activated atomic medicine” to target cancer cells having robustproliferation, a hallmark of the disease in these patients. Some of thenew molecules and their use are discussed below.

The application discloses nucleosides, or heavy nucleosides, with O¹⁸labels, and optionally also deuterium. For example, presently disclosedmodifications of thymidine comprising “heavy” thymidine (HT) are, in oneaspect, created by replacing the oxygen atom(s) attached at, forexample, the 3′ or 5′ position of the ribose structure or the oxygenatoms on the Thymine ring structure with non-radioactive oxygen-18 (¹⁸O)2-HTd for use in treatment and diagnosis.

Like thymidine, “Heavy-thymidine” is incorporated extensively into theDNA of rapidly proliferating cancer cells because its chemicalproperties are not altered relative to thymidine as far as incorporationinto DNA is concerned. Large doses of thymidine are well tolerated inhuman cancer patients with concentrations in the serum achieved in themilli-molar range. Subsequent exposure to proton irradiation activatesthe ¹⁸O by a particle-nuclear reaction that transmutates ¹⁸O into ¹⁸Fthat results in a break of the new fluorine-phosphorous bond and createsan atomic dipole at the F—C bond on the Thymidine ring. This chemicalevent destabilizes ribose-phosphate DNA back-bone and base pairing thusproduce single- and double strand breaks, clusters lesions that can leadto irreparable DNA damage and enhanced tumor cell killing. This can leadto significant deleterious effects on cancer tissue with fewconsequences on most normal tissues based on known differences inproliferation indices and the elegant dose distribution created by theproton bragg peak that totally spares “exit dose” to normal tissues.These atomic, chemical, and physical aspects result in the use of loweroverall radiation doses and significantly alter acute and late morbidityof radiotherapy. The 2-HTd analogue of thymidine has been made and theformer incorporates well into DNA (97%); preliminary investigation withour clinical 230 MeV beam has shown we can activate ¹⁸O with 5 Gye. Wenow have evidence that there is proton specific radiation sensitizationwith 2HTd plus a single dose of 8 Gye of protons.

Use of Protons and ¹⁸O Labeled Molecules for Diagnosis and Treatment ofCancer

It is disclosed herein that upon exposure to proton irradiation, theparticle-nuclear reaction transmutates ¹⁸O into ¹⁸F ((p (¹⁸O, ¹⁸F),half-life=109.8 minutes).

This new atomic state induced in the incorporated analogues can resultin a chemical break of the fluorine-phosphorous bond leading todestabilization of the ribose-phosphate DNA ladder or altered baseparing resulting in altered DNA damage repair and increase cellularkill. A simple flow diagram is provided below indicating a type oftransmutation disclosed herein:

It is also disclosed herein that ¹⁸O is converted to ¹⁸F when subjectedto a proton beam. Fluorine-18 (18F) is a fluorine radioisotope which isan important source of positrons. It has a mass of 18.0009380(6) u.Because of this short half-life, there is little chance of radiationdamage to the patient. It decays by positron emission 97% of the timeand electron capture 3% of the time. Both modes of decay yield stableoxygen-18. Fluorine-18 is an important isotope in theradiopharmaceutical industry, and is primarily synthesized intofluorodeoxyglucose (FDG) for use in positron emission tomography (PETscans). It is substituted for hydroxyl and used as a tracer in the scan.Its significance is due to both its short half-life and the emission ofpositrons when decaying. Until now, dioxaborolane chemistry has beenused to label antibodies with radioactive fluorine (¹⁸F) has beendisclosed recently, which allows for positron emission tomography (PET)imaging of cancer. Also, radioactive 2-deoxy-2-[¹⁸F]fluoro-D-glucose hasbeen used for diagnostic purposes in conjunction with PET. Disclosedherein is its use in labeling DNA, etc. and for better targeting whentreating cancer.

Without wishing to be bound by any particular theory, it washypothesized that targeted radiation sensitization with HTd analoguescan be achieved with proton treatment for the following reasons:

-   -   High tumor concentration: Daily administration of these        analogues can achieve >50% of normal serum concentrations        because they are chemically identical to their non-substituted        parent, are non-radioactive, and have both been used at        pharmacologic doses with little toxicity in humans. We calculate        this could easily achieve up to 4.8×10⁸ heavy atoms per cell,        that are assumed to be doubling every ˜24 to 48 hours.    -   Tumor proliferation and up regulated DNA synthetic pathways of        cancer cells: The hallmark of cancer is genetic alterations that        lead to excessive cellular division. This favors the utilization        of HTd analogues and their incorporation into DNA in cancer        cells compared to surrounding normal tissues.

Thus, this combination we predict will have a significant deleteriouseffect on cancer tissues with few consequences on most normal tissuesbased on cell proliferation kinetics of cancers compared to normaltissues. Furthermore, the elegant dose distribution created by protonsthat totally spares “exit dose” to normal tissues will contribute towidening the therapeutic window for patients.

Irradiation studies conducted with the proton beam: A clinical 230 MeVbeam was used to activate ¹⁸O water placed in the distal end of aspread-out Bragg peak (SOBP) delivering 5 Gye. Our data are consistentwith results of ¹⁸O proton activation data with a 160 MeV beam at the M.D. Anderson Cancer Center in Houston. Their data indicate protonactivation of ¹⁸O ranging from ˜12% in the most distal zone of the Braggpeak, to 100% activation starting at the ˜90% percent depth dose. Thesedata confirm the notion that for each nominal energy comprising a SOBPbeam, that activation, albeit somewhat inhomogeneous, can occurthroughout the irradiated volume. This is supported by the acceptedunderstanding that ¹⁸O activation peaks at 700 millibarn over the energyrange of 8 to 17 MeV, present throughout the SOBP.

A multifunctional laboratory facility was used allowing the irradiationexperiments with the proton beam in combination with the HTd analogue tobe carried out onsite and we have experience in providing precisiondosimetry. We have begun determinations of the enhancement in cellkilling with an in vitro colony assay using the glioblastoma cell lineU87-MG that has a well characterized genetic background (e.g. P53mutated or wild-type, functional or deleted PTEN and MGMT methylationstatus).

A treatment protocol used to test the use of heavy thymidine included0.125, 0.25, and 0.5 μM exposure for 96 hours. Physiologic serumconcentrations in normal and cancer patients the physiologic serumconcentration is ˜0.3-1.2 μM. The cells were irradiated with 2 and 8 Gyewith 160 MeV protons single fraction and cell death/survival wasdetermined using a colony assay and calculation of survival fraction.

The results of irradiation with 8 Gye single fraction plus 2HTd showsthere is increased cytotoxicity compared to the proton beam treatmentalone. There was about half a log increased cell kill with 0.5 microgram(a physiologic concentration in humans) of HTd exposed for 96 hours invitro (FIG. 1). There was no effect on cell survival for the sameconcentration X time exposure of regular (unsubstituted) Thymidine.These data are proof of principle for the concept of proton activatedradiation sensitization.

Various types of nucleotides can be used and labeled with heavy 180 andother atoms to be subjected to proton activation and transmutation (SeeFIG. 2 and others described in the next section). A schematic for ¹⁸Otransmutation is provided in FIG. 3 and illustrates some of the chemicalconsequences once the transmutation occurs. FIG. 4 schematicallyhighlights useful sites available for chemical modification ofpyrimidine nucleosides, using 2′ deoxyuridine as a model (based on FIG.6 from Wiebe, Brazilian Archives of Biology and Technology, 2007,50:3:445-459).

Proton Activation

Proton activated chemical transformation and its biological and otherapplications. Heavy atom labeled molecules when activated with a protonbeam are susceptible for transmutation into a radionuclide which mayresult in altering chemical bonds and its properties as a molecule. Thisphenomenon of activating an atom to transmutate to another atom wouldlikely change chemical structure and function. Shown below and in thefigures are examples of such possibilities.

Proton Activated Chemical Transformation Examples—The following flowschematic provides three examples:

Upon activation and transmutation the transient structuralperturbation/fission results in new uses, including therapeuticallyuseful biological application or chemical materials based applicationsuch as change in properties of material.

FIG. 2 provides schematically deoxyribonucleotide structures inphosphorylated forms, while FIG. 3 provides a schematic representationof ¹⁸O transmutation and the chemical consequences of the transmutation.FIG. 4 further provides a schematic representation of sites for chemicalmodification of pyrimidine nucleosides and types of modifications thatcan be made at those sites, using 2′ deoxyuridine as a model. FIG. 5,comprising FIGS. 5A and 5B, is a schematic representation of aconceptual depiction of Proton Activated Atomic Medicine. FIG. 5Ademonstrates heavy atom labeled biologically important monomericentities, their transition to oligomeric entities, the formation ofmacromolecules, and a proton beam causing transmutation and structuralchanges and leading to altered function. FIG. 5B illustrates moleculardetails depicting a nucleoside being exposed to a proton beam,atom-transmutation for example from ¹⁸O to ¹⁸F and then alteredstructure and function of biomolecules. (See FIGS. 1-5).

Proton treatment can be coupled with imaging such as immediate PET/CT.The present application encompasses, for example, tumor imaging enhancedby 5-Fluorouracil. Studies have been initiated (data not shown) usingfluorine containing compounds (including F containing nanoparticles) invitro to measure activation with sensitive sodium iodide sensor (seeFigures). There is low activity. Cho, et al. (Phys. Med. Biol. 58: 7497,2013) demonstrates the feasibility of proton-activated implantablemarkers for proton range verification using PET.

Synthesis and Use of New Molecules for Use in Proton Activated AtomicMedicine

Described below is the synthesis work of various derivatives discussedabove.

Use of ¹⁸O-heavy atom labeled biologically active small moleculesincluding natural and synthetic small molecules such as nucleosides,nucleotides, amino acids and their oligomers has been extensivelyexplored for mechanistic and diagnostic purposes for decipheringstructure function effects in peptides, proteins, DNAs, RNAs and theirfragments.¹⁻⁵ This was primarily done for stable isotope massspectrometry when analyzing the distribution and extents of its presencein cells and tissues. Previous ¹⁸O labeled compounds were sparselyutilized for other purposes such as diagnostics by imaging.⁶⁻⁷ There areseveral methods reported to for synthesis of variety of natural andsynthetic analogs of biological small molecules, in particularnucleosides and nucleotides.⁸⁻¹¹

It is proposed that synthesis of additional ¹⁸O labeled molecules, whichupon proton capture transmutate to another molecule to ensure greatertherapeutic efficacy, is encompassed by the present application and thedata disclosed herein. Disclosed herein is the first demonstration ofthe potential therapeutic application of ¹⁸O labeled nucleotides viaprotein activation (proton capture) to generate in-situ a new chemicalentity (¹⁸O labeled compound to ¹⁸F labeled compound; see FIGS. 5A and5B), which because of transmutation to another atom permits additionaltherapeutic efficacy.

Chemistry Procedures:

Preparation of Na¹⁸OH: In 50 ml plastic vial was measured 5.0 mL of H₂¹⁸O (97.4% isotopic enrichment, Isoplex, San Francisco, CA) to this wasadded very slowly and carefully dried powder NaH (230.0 mg, 90% fromAldrich, Cat. #223441) (Caution: This reaction is highly exothermic andvigorous, evolves hydrogen and may cause fire or explosion) over 3-5minutes with constant stirring in an ice-water bath (5-10° C.). Once theaddition is completed, the mixture was further stirred at roomtemperature for 30 minutes under nitrogen. This solution was used as isfor reaction with cyclic anhydride. (Water from the mixture can belyophilized under vacuo in to get powder to be stored in inertatmosphere).

Synthesis of C2-¹⁸O-Thymidine: To a solution of 2,5′-cyclic-anhydrothymidine (224.0 mg, 1 mmol, CAS #15425-09-9) was suspended in 1.0 mL ofH₂ ¹⁸O and to it was added 0.2 mL of Na¹⁸OH solution made above. Themixture was stirred at room temperature for over-night (˜12 hrs). TLCanalysis of reaction mixture indicated complete loss of starting cyclicthymidine and new compound matching the retention time with thymidinewas formed. The mixture was then carefully quenched with dil. HCl (0.1N) till slightly acidic pH˜3.0. The reaction mixture was thenlyophilized to remove all water to yield off white powder (232.0 mg,95%). ¹H-NMR (DMSO-d₆): δ 11.3 (s, 1H), 7.69 (s, 1H, ArH), 6.16 (t, J=5Hz, 1H), 5.21 (d, J=5, 1H), 5.01 (t, J=5, 1H), 4.23 (bs, 1H), 3.76 (brs,1H), 3.50-3.65 (m, 2H), 2.00-2.12 (m, 2H), 1.71 (s, 3H).: Massspectrometry: observed m/z 244, calcd. m/z 244 for C₁₀H₁₄N₂O₄ ¹⁸O.

Synthesis of C2-¹⁸O-3′epi-thymidine: To a solution of2,3′-cyclic-anhydro thymidine (224.0 mg, 1 mmol, American AdvancedScientific, College Station, Texas; Cat. #AAS-4895. CAS #15981-92-7) wassuspended in 1.0 mL of H₂ ¹⁸O and to it was added 0.2 mL of Na¹⁸OHsolution made above. The mixture was stirred at room temperature forover-night (˜12 hrs). TLC analysis of reaction mixture indicatedcomplete loss of starting cyclic thymidine and new compound matching theretention time with thymidine was formed. The mixture was then carefullyquenched with dil. HCl (0.1 N) till slightly acidic pH˜3.0. The reactionmixture was then lyophilized to remove all water to yield off whitepowder (238.0 mg, 97%). ¹H-NMR (DMSO-d₆): δ 7.70 (1H, ArH), 6.04 (d,J=10 Hz, 1H), 4.24 (bs, 1H), 3.70-3.75 (m, 3H), 3.58 (dd, J=5, 10 Hz,1H), 2.45-2.54 (m, 3H), 1.82 (d, J=14 Hz , 1H), 1.73 (s, 3H). Massspectrometry: observed m/z 244, calcd. m/z 244 for C₁₀H₁₄N₂O₄ ¹⁸O.

Synthesis of C2-¹⁸O-Uridine: To a solution of 2,5′-cyclic-anhydrouridine (210.0 mg, 1 mmol, CAS #20701-12-6) was suspended in 1.0 mL ofH₂ ¹⁸O and to it was added 0.2 mL of Na¹⁸OH solution made above. Themixture was stirred at room temperature for over-night (˜12 hrs). TLCanalysis of reaction mixture indicated complete loss of starting cyclicthymidine and new compound matching the retention time with thymidinewas formed. The mixture was then carefully quenched with dil. HCl (0.1N) till slightly acidic pH˜3.0. The reaction mixture was thenlyophilized to remove all water to yield off white powder (227 mg, 98%).¹H-NMR (DMSO-d₆): δ 7.70 (1H, ArH), 6.04 (d, J=10 Hz, 1H), 4.24 (bs,1H), 3.70-3.75 (m, 3H), 3.58 (dd, J=5, 10 Hz, 1H), 2.45-2.54 (m, 3H),1.82 (d, J=14 Hz, 1H). Mass spectrometry: observed m/z 230, calcd. m/z230 for C₉H₁₂N₂O₄ ¹⁸O.

The literature shows that ¹⁸O incorporates well into HT analogues (97%);evolving methods with this isotope currently includes biomarkers foroncology opening the possibility for the creation of other tagged heavyanalogues.

Various heavy oxygen 18 labeled thymidine (a nucleoside) derivativeshave been synthesized and are illustrated in the Figures as well asbelow. See FIG. 6 for synthetic pathways and schemes used to prepare ¹⁸Olabeled compounds. It provides some specific heavy oxygen labeledcompounds of the invention. FIG. 7 provides four examples of heavyoxygen 18 labeled thymidine derivatives along with a site formodifications. FIGS. 8-9 provide specific examples of ¹⁸O labeledcompounds of the invention.

Some of the nucleotides that were labeled with heavy oxygen are shownbelow and in the Figures.

Some singly labeled C2 and C5′-¹⁸O thymidines of the invention are (Seealso FIG. 6):

Four sites are also available on deoxyuridine for oxygen substitution.Some singly ¹⁸O labeled C2 and C5′-¹⁸O 2′-deoxyuridines are:

Some useful singly ¹⁸O labeled synthetic thymidine derivatives of theinvention labeled with heavy oxygen 18 include, but are not limited to:

wherein X is a halogen or any other group such as CH₃, OH, NH₂, OR, N₃,CH₂X.

Some singly ¹⁸O labeled thymidines of the invention include:

Some multiple ¹⁸O labeled thymidines include:

Also provided are examples of heavy oxygen 18 labeled 2′-deoxyuridinethymidine. Singly ¹⁸O labeled uridines, include, but are notlimited to:

Some uridines that are multiply labeled withO¹⁸ include:

Anhydrous thymidine was combined with ¹⁸O water (97% pure) to producethe heavy substituted analogue, 2-HTd. The synthesis of this moleculewas verified with mass spectrometry (data not shown).

See FIG. 1 for uses of the compounds.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated by reference herein intheir entirety.

Headings are included herein for reference and to aid in locatingcertain sections. These headings are not intended to limit the scope ofthe concepts described therein under, and these concepts may haveapplicability in other sections throughout the entire specification.

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention.

BIBLIOGRAPHY

1. Wang, Meiyao; Shen, Yang; Turko, Illarion V.; Nelson, Daniel C.; Li,Shuwei; 1. Determining Carbapenemase Activity with 18O Labeling andTargeted Mass Spectrometry, Analytical Chemistry (Washington, DC, UnitedStates) (2013), 85(22), 11014-11019.

2. Bergmann, Dominik; Huebner, Florian; Humpf, Hans-Ulrich; StableIsotope Dilution Analysis of Small Molecules with Carboxylic AcidFunctions Using 18O Labeling for HPLC-ESI-MS/MS: Analysis of FumonisinB 1. Journal of Agricultural and Food Chemistry (2013), 61(33),7904-7908.

3. Moore, Ronald J.; Schepmoes, Athena A.; Xiao, Wenzhong; Moldawer,Lyle L.; et al; Plasma Proteome Response to Severe Burn Injury Revealedby 18O-Labeled “Universal” Reference-Based Quantitative Proteomics.Journal of Proteome Research (2010), 9(9), 4779-4789.

4. Mori, Masaru; Abe, Kohei; Yamaguchi, Hiroaki; Goto, Junichi; Shimada,Miki; Mano, Nariyasu, Production of 180-Single Labeled Peptide Fragmentsduring Trypsin Digestion of Proteins for Quantitative Proteomics UsingnanoLC-ESI-MS/MS. Journal of Proteome Research (2010), 9(7), 3741-3749.

5. Kolodziejska-Huben, Magdalena; Kaminski, Zbigniew; Paneth,PiotrPreparation of 180-labeled nicotinamide. Journal of LabelledCompounds & Radiopharmaceuticals (2002), 45(12), 1005-1010.

6. Hamasaki, Tomohiro; Matsumoto, Takahiro; Sakamoto, Naoya; Shimahara,Akiko; Kato, Shiori; Yoshitake, Ayumi; Utsunomiya, Ayumi; Yurimoto,Hisayoshi; Gabazza, Esteban C.; Ohgi, Tadaaki; Synthesis of 18O-labeledRNA for application to kinetic studies and imaging. Nucleic AcidsResearch (2013), 41(12), e126.

7. Xiao, Bin; Li, You; Zhou, Jianyue; Jiang, Yongyue; Liu, Yan; Qin,Chuanjiang; Method and apparatus for preparation of oxygen 18 labeledwater as nuclear medicine diagnostic reagent. Faming Zhuanli Shenqing(2009), CN 101575086 A 20091111.

8. H. Follmann and H. P. C. Hogenkamp, The synthesis of ribose and ofadenine nucleotides containing oxygen-18. The Journal of AmericanChemical Society, 92:3, 1970.

9. Schram, Karl H.; Ratcliff, Steven; Neenan, John; Oxygen-18-labelednucleosides. 1. A general method for the synthesis of specificallylabeled pyrimidine deoxyribosides. Journal of Labelled Compounds andRadiopharmaceuticals (1982), 19(3), 399-404.

10. Solsten, R. Thomas; McCloskey, James A.; Schram, Karl H.Oxygen-18-labeled nucleosides. 2. A general method for the synthesis ofspecifically labeled pyrimidine ribonucleosides. Nucleosides &Nucleotides (1982), 1(1), 57-64.

11. Schubert, Ernst M.; Schram, Karl H; Oxygen-18-labeled nucleosides.3. Preparation and mass spectrometric evaluation of 1802-labeled1-(β-D-arabinofuranosyl)cytosine and -uracil. Journal of LabelledCompounds and Radiopharmaceuticals (1982), 19(8), 929-35.

12. Wiebe, L., Applications of Nucleoside-based Molecular Probes for thein vivo Assessment of Tumour Biochemistry using Positron EmissionTomography (PET). Brazilian Archives of Biology and Technology (2007),50:3:445-459.

What is claimed is:
 1. A compound of the formula:

wherein: X is selected from the group consisting of H, CH₃, halogen,CF₃, OH, NH₂, OR, N₃, and CH₂X; R is selected from the group consistingof alkyl, alkene, alkyne, and aryl; and said compound optionallycomprises at least one additional ¹⁸O.
 2. The compound of claim 1,wherein said compound comprises at least one additional ¹⁸O. 3.(canceled)
 4. The compound of claim 2, wherein three oxygens are ¹⁸O. 5.The compound of claim 2, wherein four oxygens are ¹⁸O.
 6. The compoundof claim 1, wherein said compound is deuterated.
 7. The compound ofclaim 1, wherein said compound is selected from the group consisting of:


8. (canceled)
 9. (canceled)
 10. A method of treating a tumor usingproton beam therapy in a subject in need thereof, said method comprisingadministering to said subject a pharmaceutical composition comprising aneffective amount of a compound of claim 1 and subjecting said tumor toproton beam therapy, thereby treating a tumor using proton beam therapy.11. The method of claim 10, wherein said compound is selected from thegroup consisting of:


12. The method of claim 10, wherein said compound is deuterated.
 13. Themethod of claim 10, wherein said tumor is imaged using positron emissiontomography (PET) or computerized tomography (CT).
 14. The method ofclaim 10, wherein an ¹⁸O of said compound is transmutated to ¹⁸F uponexposure to said proton beam.
 15. (canceled)
 16. The method of claim 10,wherein said treatment is used in combination with another treatmentselected from conventional x-ray irradiation, chemotherapy, and surgery.17. (canceled)
 18. The method of claim 10, wherein said proton beamtherapy comprises a total dose of about 50 to about 150 Gy. 19.(canceled)
 20. (canceled)
 21. (canceled)
 22. A method of killing aproliferating cancer cell, said method comprising contacting saidproliferating cancer cell with an effective amount of a compound ofclaim 1, allowing said effective amount of said compound to incorporateinto the DNA of said proliferating cancer cell, and subjecting saidproliferating cancer cell to proton beam therapy.
 23. (canceled)
 24. Themethod of claim 10, wherein said tumor is selected from the groupconsisting of brain, breast, bone, gastrointestinal tract, prostate, andpediatric tumors.
 25. (canceled)
 26. A pharmaceutical compositioncomprising a pharmaceutically-acceptable carrier and a compound ofclaim
 1. 27. (canceled)
 28. A kit comprising an effective amount of atleast one compound of claim 1, a pharmaceutically-acceptable carrier, anapplicator, and an instructional material for the use thereof.